Molecular epidemiology of Enterocytozoon bieneusi from foxes and raccoon dogs in the Henan and Hebei provinces in China

Background Enterocytozoon bieneusi is a zoonotic pathogen widely distributed in animals and humans. It can cause diarrhea and even death in immunocompromised hosts. Approximately 800 internal transcribed spacer (ITS) genotypes have been identified in E. bieneusi. Farmed foxes and raccoon dogs are closely associated to humans and might be the reservoir of E. bieneusi which is known to have zoonotic potential. However, there are only a few studies about E. bieneusi genotype identification and epidemiological survey in foxes and raccoon dogs in Henan and Hebei province. Thus, the present study investigated the infection rates and genotypes of E. bieneusi in farmed foxes and raccoon dogs in the Henan and Hebei provinces. Result A total of 704 and 884 fecal specimens were collected from foxes and raccoon dogs, respectively. Nested PCR was conducted based on ITS of ribosomal RNA (rRNA), and then multilocus sequence typing (MLST) was conducted to analyze the genotypes. The result showed that infection rates of E. bieneusi in foxes and raccoon dogs were 18.32% and 5.54%, respectively. Ten E. bieneusi genotypes with zoonotic potential (NCF2, NCF3, D, EbpC, CHN-DC1, SCF2, CHN-F1, Type IV, BEB4, and BEB6) were identified in foxes and raccoon dogs. Totally 178 ITS-positive DNA specimens were identified from foxes and raccoon dogs and these specimens were then subjected to MLST analysis. In the MLST analysis, 12, 2, 7 and 8 genotypes were identified in at the mini-/ micro-satellite loci MS1, MS3, MS4 and MS7, respectively. A total of 14 multilocus genotypes were generated using ClustalX 2.1 software. Overall, the present study evaluated the infection of E. bieneusi in foxes and raccoon dogs in the Henan and Hebei province, and investigated the zoonotic potential of the E. bieneusi in foxes and raccoon dogs. Conclusions These findings expand the geographic distribution information of E. bieneusi’ host in China and was helpful in preventing against the infection of E. bieneusi with zoonotic potential in foxes and raccoon dogs. Supplementary Information The online version contains supplementary material available at 10.1186/s12917-024-03883-6.


Background
Microsporidia are obligate intracellular parasites with hosts ranging from protists to mammals [1].More than 200 genera and approximately 1,500 species of microsporidia have been identified, and 17 species causes the infection of human beings.E. bieneusi is responsible for more than 90% cases of human microsporidiosis infection [1][2][3][4][5][6].Since its discovery in an acquired immunedeficiency syndrome patient in 1985, many genotypes have been identified [7,8].E. bieneusi could induce diarrhea or even death of the patient, but most of the patients infected with E. bieneusi only showed slightly dysbiosis or disruption of nutrient absorption [2,9,10].
More than 800 genotypes of E. bieneusi have been identified using polymorphism analysis of the internal transcribed spacer (ITS) region of the rRNA gene which belongs to 13 phylogenetic groups [2,4,11,12].More than 310 genotypes are included in Group 1 which are believed to infect both human and animals.BEB4, BEB6, I, and J are the dominant genotype in Group 2 which are found in ruminants, non-ruminant animals, and humans.Genotypes in Group 3-13 infect animals and showed little effect on public health [13].Different groups of E. bieneusi genotypes display diverse zoonotic potential and host specificity [4].Group 1 (e.g., genotype D, Type IV, and EbpC) is the largest group of E. bieneusi genotypes and can infect different kinds of animals with high adaptability to the environment [4,14].Most of Group2 members of E. bieneusi, e.g., genotype I, J, BEB4, and BEB6 are the most common genotypes of E. bieneusi identified in sheep, goats, cattle, and deer [4,6].Most genotypes of E. bieneusi in groups 3-11 have a limited host range and thus pose a minor or unknown public health threat [4].Nevertheless, the ITS genotyping method cannot fully reflect the genetic characteristics of E. bieneusi as it represents only a limited portion of the E. bieneusi genome (total length about 6 Mb) [15][16][17].Multilocus sequence typing (MLST) is more discriminatory than ITS genotyping method by taking genetic polymorphisms of four mini-and microsatellites into account [8,16,18].A higher genetic diversity was identified in E. bieneusi isolated from humans and animals using MLST analysis and several genetically isolating subgroups were formed within the ITS group 1 owing to their characteristics [19][20][21].
However, there are only a few epidemiological studies on E. bieneusi in foxes and raccoon dogs in captivity worldwide.Thus, to further understand the genetic diversity of E. bieneusi in foxes and raccoon dogs, obtain geographic information, and compare the infection rates of E. bieneusi in different regions, the present study analyzed the infection rates and genotypes of E. bieneusi in farmed foxes and raccoon dogs in the Henan and Hebei provinces using MLST.

Results and discussion
As shown in Table 1, a total of 178 E. bieneusi-positive samples (11.21%, 95% CI: 9.66-12.76)were identified via nested PCR based on the ITS locus in 1588 fecal samples from foxes and raccoon dogs and the total infection rate of E. bieneusi was similar with the total infection rate of E. bieneusi in farmed blue foxes and raccoon dogs was 12.6% in the Heilongjiang and Jilin Province [27], while was higher relative to the total infection rate of farmed blue foxes and raccoon dogs in Xinjiang China (2.7%) [32].This indicated that prevalence of E. bieneusi was associated with geographic distribution of the animals.The infection rates of E. bieneusi were 8.65% (84/971), 7.81% (21/269), and 20.98% (73/348) in samples from Xinxiang city, Hebi city, and Changli city.The infection rate in samples from Changli city was significantly higher than that from Xinxiang city and Hebi city (P < 0.01).The infection rate in foxes was 18.32% (129/704), which was significantly higher relative to that in raccoon dogs (5.54%, 49/884) (P < 0.01).The infection rate of E. bieneusi in foxes and raccoon dogs in the present study was similar with previous findings in which E. bieneusi was detected in 16.4% (18/110) farmed blue foxes and 4.1% (2/49) raccoon dogs [27].The infection rate in preweaned foxes (3.51%, 95% CI: 17.84-24.70)was lower than that in young (20.00%, 95% CI: 7.04-32.96)and adult foxes (21.27%, 95% CI: 17.84-24.70).The lower infection rate in pre-weaned foxes observed in the present study might be associated with the immune status and the antibodies contained in the colostrum, but the mechanism should be elucidated further.The infection rate in male foxes was slightly lower than that in female foxes which was different from previous study in which they found no significant difference in the infection rate of E. bieneusi between male and female foxes [33].The infection rate in male raccoon dogs (8.17%, 95% CI: 5.67-10.67)was higher than that in female raccoon dogs (2.63%, 95% CI: 1.09-4.16),and this finding is in line with the results of previous studies [24,26].The differences observed in the infection rate of E. bieneusi in foxes and raccoon dogs of different gender in the present study maybe associated with sample size, different animal husbandry practice and animal welfare.Study demonstrated that no effective therapeutic method was available for the treatment of E. bieneusi [34].This might be the reason why no significant difference in the infection rate between dewormed (dewormed with Avermectin) and non-dewormed farm animals was observed in the present study.
As shown in Table 2, ten genotypes (NCF2, NCF3, D, EbpC, CHN-DC1, SCF2, CHN-F1, Type IV, BEB4, and BEB6) were identified by sequencing in the present study, among which genotype NCF2 was the dominant one, and all genotypes identified in the present study were zoonotic [34].The genotypes NCF2, NCF3, D, CHN-DC1, and SCF2 has been identified in foxes previously [5,22,25,[27][28][29][30][31], but the genotypes SCF2, CHN-F1, and BEB6 were first identified in foxes in the present study.Raccoon dogs has been reported to be infected with genotypes NCF2, D, and Type IV previously [11,[22][23][24][25][26][27], but the genotypes NCF-3, EbpC, SCF-2, BEB4, and BEB6 were first identified in raccoon dogs in the present study (see Table 2).Among them, genotypes BEB4 and BEB6 belong to the ITS group 2 which have not been reported to be found in foxes and raccoon dogs.Previous studies identified genotype BEB4 in cattle, yaks, pigs, humans, and non-human primates [4], while genotype BEB6 was identified in cattle, sheep, goats, and humans [35,36].Thus, we hypothesized that genotypes BEB4 and BEB6 identified in foxes and raccoon dogs in the present study may be transmitted from cattle, because all the genotypes BEB4 and BEB6 identified in the present study were from the same farm which is close to a cattle farm.This transmission may be due to the contamination of the raw water by the feces of infected cattle in the farm nearby, but the prevalence of E. bieneusi in the cattle farm and raw water was not evaluated in the present study, therefore further study is still needed to clarify our hypothesis.Phylogenetic analysis of the ITS loci showed that genotypes NCF2, NCF3, D, EbpC, CHN-DC1, SCF2, CHN-F1, and Type IV clustered into ITS group 1.Among them, genotypes EbpC, Type IV, and D are the most common genotypes of E. bieneusi that affect not only humans but also livestock and wild animals worldwide [4].Although the genotypes BEB4 and BEB6 belong to ITS group 2 whose hosts are mostly ruminants, they may transmit to other hosts and lead to the infection of human beings [34].Overall, these findings suggest that foxes and raccoon dogs may be potential sources of E. bieneusi infection in humans and other animals (Fig. 1).
The phylogenetic analysis of microsatellite and microsatellite loci revealed that most of the E. bieneusi isolates from foxes and raccoon dogs were clustered together with the isolates from pigs and showed a close genetic match (Fig. 2).A few E. bieneusi isolates found in the present study were closest matched with the isolates from bear, Vicugna pacos, and squirrel and non from humans (Fig. 2), which is consistent with results of a previous study [18].
The findings of in the present study enrich the knowledge on the genetic diversity of E. bieneusi in foxes and raccoon dogs and performed the epidemiological investigation of E. bieneusi in foxes and raccoon dogs in the Henan Province and Hebei Province which has not been done in China.Currently, little information is available about the epidemiology of E. bieneusi in wild foxes and raccoon dogs; thus, wild species should be the focused in future studies.

Conclusion
In the present study, differences in the infection rates of E. bieneusi in foxes and raccoon dogs were assessed by region, breed, age, sex, and deworming condition.Ten zoonotic E. bieneusi genotypes (i.e., NCF2, NCF3, D, EbpC, CHN-DC1, SCF2, CHN-F1, Type IV, BEB4, and BEB6) were identified, and a total of 14 MLGs were formed.Findings of the present study are benefit for the control and prevention of E. bieneusi infection in foxes and raccoon dogs.

Sample collection
Fresh fecal samples were collected from the rectum of foxes and raccoon dogs using disposable chlorinated polyethylene (CPE) gloves.Then specimens were placed in an ice-cold container and transported to the laboratory immediately.Half of the fecal samples were stored at 4 °C for DNA extraction, and the remaining samples were soaked in 2.5% potassium dichromate and stored at -20℃.A total of 1588 samples were collected between June and December 2020 from eight farms in Henan and Hebei province and full name of the farms were listed in table S1 in the supplementary file.The detailed information regarding sample collection was presented in Table 1.

DNA extraction
Genomic DNA was extracted using the Stool DNA Kit (Omega Bio-Tek Inc., Norcross, GA, USA) according to the manufacturer's instruction and the isolated DNA was stored at -20℃.

PCR amplification and MLST
Infection of E. bieneusi were evaluated by nest PCR assay based on ITS locus, and the primers used in the present study has been described in our previous study [37].The ITS-positive samples were selected based on ITS genotype, region, breed, age, and sex, and then were subjected to MLST analysis at the MS1, MS3, MS4, and MS7 loci.
The primers and annealing temperatures used in the MLST analysis of the present study were described previously [14].The secondary PCR products were visualized by 1.5% agarose gel electrophoresis (containing 1 × 10 − 5 DNA Green).

Sequencing and phylogenetic analysis
The ITS positive secondary PCR products were sent to SinoGenoMax Biotechnology Co., Ltd.(Beijing, China) for sequencing and sequences obtained were aligned with reference sequences downloaded from the Gen-Bank (http://blast.ncbi.nlm.nih.gov) using Clustal X 2.13 (http://www.clustal.org/)to confirm different species or genotypes.
To determine the phylogenetic relationships among the detected genotypes, neighbor-joining trees were constructed using the MEGA VII program (www.megasoftware.net) based on evolutionary distances calculated with the Kimura 2-parameter model.The reliability of

Fig. 1
Fig. 1 Phylogenetic relationships among E. bieneusi isolates inferred with a neighbor-joining analysis based on the ITS nucleotide sequences.The reliability of cluster formation was assessed by the bootstrap analysis with 1000 replicates, and the values generated greater than 50% are shown beside the nodes.The known ITS genotypes identified in the present study are indicated by black triangles

Fig. 2
Fig. 2 Phylogenetic relationships among E. bieneusi isolates inferred with a neighbor-joining analysis based on MS1, MS3, MS4 and MS7 locus, respectively.The reliability of cluster formation was assessed by the bootstrap analysis with 1000 replicates, and the values generated greater than 50% are shown beside the nodes.The types identified in the present study are indicated by black triangles

Table 1
Factors associated with the prevalence of E. bieneusi in farmed foxes and raccoon dogs in the Henan and Hebei provinces OR, odds ratio: CI, confidence interval.a Samples from foxes.b Samples from raccoon dogs

Table 2
Genotypes of E. bieneusi in farmed foxes and raccoon dogs in the Henan and Hebei provinces

Table 3
Multilocus genotypes of 14 E. bieneusi isolates from foxes and raccoon dogs